1. Field of the Invention
This invention relates to sperm-specific protein kinase A anchoring proteins from mammalian species and the genes encoding such proteins. Specifically, the invention relates to the isolation of nucleic acid encoding sperm-specific protein kinase A anchoring proteins from mammalian species, and the production of homogenous preparations of such proteins using recombinant genetic techniques. The invention also relates to the preparation of analytic tools such as polyclonal antisera and monoclonal antibodies specific for said sperm-specific protein kinase A anchoring proteins. Importantly, the invention is related to high throughput screening of compounds that can disrupt binding between protein kinase A and the sperm-specific protein kinase A anchoring proteins of the invention, or that inhibit or prevent expression of the sperm-specific protein kinase A anchoring proteins in sperm, spermatids or progenitor cells thereof, for development of effective contraceptives, and provides antisense oligonucleotides, agents and compounds detected using the screening methods of the invention as reversible, male contraceptive agents.
2. Background of the Invention
Signal transduction enzymes such as protein kinases and phosphatases play pivotal roles in mediating cellular responses to a wide variety of stimuli. Cyclic AMP (cAMP) mediates the motility of sperm and a variety of other ciliated cells (Satir, 1985, Molec. Cell Biol. 4: 1-46n Tash, 1989, Cell Motil. Cytoskel. 14: 332-339; Bedford and Hoskins 1990, in MARSHALL""S PHYSIOLOGY OF REPRODUCTION, (Lamming, ed.), Churchill Livingstone: New York, p.t379). Increases in the level of this nucleotide are associated with development of sperm motility in the epididymis (Bedford and Hoskins, ibid.; Hoskins et al., 1974, J. Reprod. Fertil. 537: 131-133). Cell-permeant cAMP analogs, cAMP phosphodiesterase inhibitors, and adenylyl cyclase activators all stimulate motility of sperm from several species (Garbers et al., 1972, Biol. Reprod. 7: 132; Garbers et al., 1978, Adv. Cyclic Nucleotide Res. 9: 583-595). Flagellar activity in sperm has been shown to be regulated by cAMP (Tash, ibid.; Garbers and Kopf, 1980, in ADVANCES IN CYCLIC NUCLEOTIDE RESEARCH, (Greengard and Robison, eds.), Vol. 13, Raven Press: New York, pp. 252-306; Lindemann and Kanous, 1989, Arch. Androl. 23: 1-22; Tash and Means, 1-988, Prog. Clin. Biol. Res. 267: 335-355).
The cAMP-dependent protein kinase (PKA) is a ubiquitous, multifunctional enzyme involved in the regulation of a diverse array of cellular events. PKA holoenzyme consists of four subunits, two catalytic (C) and two regulatory (R). In the absence of cAMP, the regulatory subunits keep the catalytic subunits inactive. Cyclic AMP binding to the regulatory subunit promotes dissociation from and activation of the catalytic subunits. There are two R subunit classes, RI and RII, which form the type I and type II holoenzymes, respectively. Type II PKA is present in all cells, whereas the tissue distribution of type I PKA is more restricted. Subcellular localization of PKA is directed through the regulatory (R) subunit. Recent studies have shown that PKA is anchored at specific subcellular sites through the interaction of its regulatory subunit with A-kinase anchoring proteins (AKAPs) (Carr et al., 1991, J. Biol. Chem. 266: 14188-14192; Rubin, 1994, Biochim. Biophys. Acta 1224: 467-479; Dell""Aqua and Scott, 1997, J. Biol. Chem. 272: 12881-4; Faux et al., 1996, Cell 85: 9-12; Pawson et al., 1997, Science 278: 2075-2080).
A number of AKAPs have been cloned and biochemically characterized (Dell""Aqua and Scott, ibid.). All AKAPs contain a common structural motif that binds with nanomolar (nM) affinity to the regulatory subunit of PKA (Carr et al., 1991, ibid.; Carr et al., 1992, J. Biol. Chem. 267: 13376-13382). Certain of the anchoring proteins that are capable of binding both RI and RII have been labeled dual (D)-AKAPs (Huang et al., 1997, J. Biol. Chem. 272: 8057-8064; Huang et al., 1997, Proc. Natl. Acad. Sci. USA 94: 11184-11189). Since PKA has broad substrate specificity, presumably one of the primary functions of PKA anchoring is to spatially restrict its action, thus providing specificity of function.
Because cAMP analogs and agents that increase intracellular cAMP are potent stimulators of sperm motility, the role of PKA and AKAP subcellular localization of PKA was expected by the inventors to be important for understanding and modulating sperm motility. The highly polarized sperm cell is particularly well suited for investigating the structure and function of PKA anchoring proteins. Differentiated sperm have a number of district subcellular structures. The microtubular apparatus in mammalian sperm is surrounded by district cellular organelles such as the outer dense fibers and fibrous sheath. The cytoplasmic volume of the sperm cell is considerably lower than that of most somatic cells. Free diffusion of plasma membrane proteins is apparently restricted to three distinct domains, defined by the sperm head, midpiece and tail regions (Friend, 1982, J. Cell Biol. 93: 243-249). This unique compartmentalization of the sperm cell made it an excellent model system to study the role of targeting and anchoring of PKA and other enzymes in regulating cell function.
Regulation of sperm motility also provides an important avenue for developing a new generation of contraceptive agents. In the past, contraception has relied on barrier methods (such as condoms) and in regulating a female""s capacity to be impregnated or the fertilized zygote to attach to the uterus (examples include the intrauterine device and xe2x80x9cthe Pillxe2x80x9d). Regulation of cAMP-stimulated sperm motility by targeting the PKA/AKAP interaction provides for the first time a male-specific route of contraceptive intervention.
There thus remains a need in this art for specific, effective, reversible, reliable male contraceptive with minimal physiological or systemic side effects. As disclosed herein, one particular PKA anchoring protein, termed AKAP110, is uniquely expressed in sperm, making it possible that therapies which interfere with or prevent expression of this protein could be used as a contraceptive. One example of such an approach is to use antisense oligonucleotides to inhibit or prevent AKAP110 gene expression in developing sperm or spermatids, which have been shown to express this protein. Antisense-based therapeutics are already being used in humans, and it is likely that systemic administration of antisense reagents will be achieved in the near future. Antisense approaches to male contraception have clear advantages over other methods, such as hormonal or immunological methods, such as being completely reversible and in having a likelihood of fewer and less physiologically significant side effects. Other examples of novel contraceptive approaches is the use of peptides or small molecules that directly interfere with the AKAP110: PKA binding interaction in the absence of threshold levels of cAMP.
The present invention relates to the cloning, expression and functional characterization of mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of about 110 kildaltons, termed xe2x80x9cAKAP110xe2x80x9d herein.
This invention specifically provides isolated nucleic acid encoding sperm-specific protein kinase A anchoring protein from human, bovine and murine male germ cells, recombinant expression constructs comprising said nucleic acid, and cells transformed with such recombinant expression constructs and producing the sperm-specific protein kinase A anchoring protein thereby. The invention also provides homogenous preparations of sperm-specific protein kinase is A anchoring protein, and antibodies raised against and that immunologically recognize said protein. The invention also provides methods for producing said homogeneous preparation of the sperm-specific protein kinase A anchoring proteins of the invention using said cells transformed with the recombinant expression constructs of the invention. The invention advantageously provides methods for screening compounds for the capacity to disrupt binding between the sperm-specific protein kinase A anchoring protein of the invention and protein kinase A regulatory subunits, and methods for disrupting, inhibiting or preventing expression of this protein in developing sperm cells, spermatids or progenitor cells thereof, in order to provide male-specific contraception agents. Methods of male contraception, most preferably and advantageously readily reversible methods of male contraception, and methods for the prevention of pregnancy are provided by the use of compounds that disrupt, inhibit or prevent expression of the AKAP110 protein of the invention, whereby such compounds are detected or characterized using the methods of the invention. Advantageously, the invention also provides agents, preferably pharmaceutical agents, for effecting male contraception, most preferably comprising antisense oligonucleotides for inhibiting or preventing expression of AKAP1 10 in sperm cells, spermatids and progenitors thereof, and peptides and small molecules for disrupting AKAP 110/PKA RII binding in said cells.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.